Anammox, an energy-efficient bioprocess, is gaining growing attention. However, its widespread adoption confronts challenges due to residual nitrate and unstable nitrite supply, especially during seasonal temperature fluctuations. To address this, we developed an innovative Anammox-mediate biotechnology, incorporating a double nitrite autotrophic shunt: partial nitrification (PN/A) and sulfur-driven partial denitrification (SPDN/A). Remarkably, a nitrogen removal efficiency was realized over 183 days, encompassing a significant seasonal temperature decrease (from 25.3 degrees C to 13.2 degrees C). Functional gene analysis revealed Anammox spontaneously downregulated functional gene expression to allocate core metabolic energy for essential cellular processes to-wards temperature plunge condition. Interestingly, as seasonal temperature decreased, nitrogen removal contribution of Anammox in SPDN/A exhibited a noticeable upward tendency from 66.4 % to 86.7 %. Enriched Thiobacillus and decreased NO2 reductase gene expression in SPDN/A ensured sustainable nitrite supply for Anammox process, promoting superiority of Candidatus Kuenenia to 7.3 %. However, Anammox groups decreased significantly in PN/A. Divergence in responses towards temperature fluctuation credibly concluded Anammox's correlation with temperature is not necessarily positive or parallel. Instead, stable provision of NO2 is primary determinant. Comprehensive analysis, integrating functional gene transcription, microbiota synergetic interactions, and kinetics underscores PN/A-SPDN/A's potential for reliable nitrogen removal, offering a sustainable solution subject to seasonal temperature fluctuation.
